Project Summary/Abstract The 3D-structures of amyloid-? fibrils have been visualized extensively in vitro, but there has yet to be a direct measurement of amyloid-? misfolding and conformer composition in vivo in Alzheimer disease (AD) patients. Amyloid-? and MAPT proteoforms aggregate in the extracellular space and in neurons, respectively, and the appearance of MAPT and amyloid-? fibrillary protein aggregates has been tightly linked to the onset of clinical symptoms in sporadic AD. While different MAPT and amyloid-? protein conformers can polymerize in distinct filaments, as visualized by ssNMR and cryo-EM in vitro, it remains unclear which protein conformers contribute relatively to misfolded MAPT and amyloid-? in vivo. Here we measure protein conformers that are associated with AD with Covalent Protein Painting, which is a mass spectrometry-based approach to analyze structural aspects of a proteome. Using CPP we covalently modify lysine residues of MAPT and amyloid-? conformers with isotope-encoded methyl moieties in vivo and quantify their relative contributions with mass spectrometry. Because different MAPT and amyloid-? conformers harbor distinct solvent exposures at select lysine residues, results obtained with CPP reflect the relative contribution of each different protein conformer in vivo. CPP enables a high throughput analysis of many proteins and readily provides a quantitation of the diversity of protein conformers and its variability between AD patients. We propose to apply CPP to determine the conformer space of MAPT and amyloid-? proteoforms that are present in brain tissue, cerebrospinal fluid and blood plasma of AD patients and of healthy, age-matched controls. We will determine at which earliest point in a patient's age misfolded amyloid-? and MAPT can be detected. In addition, CPP will be used to infer which additional proteins in which conformation interact specifically with amyloid-? and MAPT that are misfolded. By establishing conformer-specific interactomes, we will identify specific sites in proteins that might be amenable to drug targeting. Furthermore, CPP is applied to scan the proteome of brain tissue and blood plasma in order to discover changes in protein conformation that lead to or accompany a condition that is permissive to protein misfolding. CPP provides a sensitive, conformer-specific assay that quantifies the proportion of misfolded amyloid-? and MAPT in tissue and holds promise to allow for an earlier detection of AD onset and progression. Performing a conformational analysis of amyloid-? and MAPT misfolding with CPP is a step towards the development of a faster drug screening method in AD models in vivo.